Thermolabile Exonucleases

ABSTRACT

The invention provides an exonuclease or an enzymatically active fragment thereof, said exonuclease having the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which is at least about 50% identical thereto, wherein said exonuclease or enzymatically active fragment thereof (i) is substantially irreversibly inactivated by heating at a temperature of about 55° C. for 10 minutes in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl 2 ; (ii) is substantially specific for single stranded DNA; and (iii) has a 3′-5′ exonuclease activity. The invention further provides a method of removing single stranded DNA from a sample, a method of nucleic acid amplification, a method of reverse transcription and a method of nucleic acid sequence analysis in which the exonuclease or enzymatically active fragment thereof is used. The invention still further provides nucleic acids encoding said exonuclease or an enzymatically active fragment thereof and kits or compositions comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/504,507, filed 16 Feb. 2017, which in turn is a nationalstage filing under 35 U.S.C. § 371 of PCT/EP2015/001703, filed on 19Aug. 2015, which in turn claims the benefit of priority to and thebenefit of GB Application No. 1414745.8, filed 19 Aug. 2014. Eachapplication is incorporated herein by reference in its entirety.

SEQUENCE LISTING SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled3403-128SequenceListing.txt, created on 30 Jul. 2010, and is 68 kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

The present invention relates to thermolabile exonucleases and the useof the same to remove single stranded DNA from samples containingbiological molecules, in particular the products of nucleic acidamplification or reverse transcription reactions (e.g. double strandedDNA, RNA and DNA:RNA duplexes). The invention may therefore be viewedparticularly as relating to the refinement of samples containing doublestranded DNA, RNA and/or DNA:RNA duplexes by removing single strandedDNA. More specifically the invention relates to the removal of excessoligodeoxyribonucleotide primers from the products of nucleic acidamplification or reverse transcription reactions. Removal of excessoligodeoxyribonucleotide primers from the products of nucleic acidamplification reactions may enhance the downstream sequence analysis ofsuch products as interference from excess oligodeoxyribonucleotideprimers is reduced. The present invention therefore further relates tomethods for optimising the sequence analysis of the products of anucleic acid amplification or reverse transcription reactions, e.g. bynucleic acid sequencing or oligonucleotide probe arrays. Methods foroptimising the sequence analysis of nucleic acids that have not beenobtained from a nucleic acid amplification reaction are also provided.

Nucleases are enzymes that break the phosphodiester bonds in thesugar-phosphate backbone of DNA or RNA polymers. Nucleases are a verydiverse group of enzymes. The mode of action can be either highlyspecific or very general depending upon their target function. Nucleasesmay prefer single stranded (ss) polymers or double stranded (ds)polymers. Some nucleases cleave at specific nucleotide sequences (e.g.restriction endonucleases), whereas others cleave at positions in thepolymers independent of nucleotide sequence. In the cell, nucleases havea variety of functions and are involved in DNA replication,recombination, mutation, transcription and repair in addition tobreaking down redundant pieces of RNA and DNA. Nucleases may also servea role in host defence mechanisms.

All nucleases may be divided into three major classes based on whethertheir catalytic mechanism involves two, one or no metal ions (thetwo-metal-ion dependent, one-metal-ion-dependent and metal independentnuclease superfamilies). Each of these classes includes many differentfamilies and superfamilies. For more details about nucleaseclassification see Yang, W., 2011, “Nucleases: diversity of structure,function and mechanism.” Q Rev Biophys 44(1): 1-93.

Based on substrate preference nucleases may be classified asdeoxyribonucleases (DNases) or ribonucleases (RNases), i.e. enzymes thatcleave the phosphodiester bonds of either DNA or RNA, respectively.Based on the positions of the cleaved bonds within the DNA or RNApolymers, nucleases may be further classified as endonucleases orexonucleases. The endonucleases cleave phosphodiester bonds of DNA orRNA at positions within the polymer, whereas exonucleases are involvedin trimming the ends of RNA and DNA polymers, cleaving the outermostphosphodiester bond in a chain. Exonucleases can be further divided intotwo groups by the 5′ to 3′ versus 3′ to 5′ polarity. Nucleases may alsodisplay specificity between single- and double-stranded nucleic acids. Aparticular nuclease may be strictly single-strand-specific,single-strand-preferential, strictly double-strand-specific,double-strand-preferential or a nuclease that cleaves both.

A number of exonuclease enzymes are known, and among them, theEscherichia coli enzymes are the most well-characterised ones. A commonfeature for exonucleases is their high processivity degrading up to a1000 nucleotides in a single binding event releasing mononucleotides.Exonuclease I (ExoI), Exonuclease VII (ExoVII) and RecJ exonuclease areall reported to be ssDNA-specific exonucleases involved in DNA repair.However, their polarity of action is different. ExoI possesses 3′ to 5′exonuclease activity while RecJ possesses only 5′ to 3′ activity. Incontrast to the other exonucleases, ExoVII possesses both 5′ to 3′ and3′ to 5′ exonuclease activities.

The determination of the sequence of nucleotides in nucleic acids, e.g.DNA and RNA, has become an important goal in modern molecular biology.By analysing such sequences information on the source of the nucleicacid can be obtained. For instance, the nucleotide sequence of certainevolutionarily variable genetic elements can provide an indication as tothe identity of the organism from which it is derived. Accordingly,detecting nucleic acid carrying a nucleotide sequence characteristic ofa particular microorganism in a sample can indicate the presence of themicroorganism in the sample or even quantify the amounts of suchorganisms in the sample. Sequence analysis can also assist in thetaxonomic classification of higher organisms, which may be important intechnical fields such as agriculture and veterinary science. In humans,nucleotide sequence analysis can identify individuals and their lineage,thus having forensic applications, and can identify medically orphysiologically relevant genotypes, e.g. mutations. The sequencing ofthe RNA transcripts of a target cell or group of cells (e.g. a tissue, atumour or a culture) can yield information on the transcriptome of thetarget, which in turn can have numerous applications in the medical andscientific fields. Nucleic acid based identity tags carrying uniquenucleotide sequences are also available, the detection of which requiresthe analysis of the nucleotide sequence of the tag. In otherrepresentative applications the skilled person may wish to ascertain thenucleotide sequence of a nucleic acid with which he/she is working,perhaps either to confirm a manipulation has succeeded or to understandthe make-up of a novel molecule.

Nucleic acid sequence analysis may take the form of a sequencingtechnique. The Sanger dideoxynucleotide sequencing method is awell-known and widely used technique for sequencing nucleic acids.However more recently the so-called “next generation” or “secondgeneration” sequencing approaches (in reference to the Sangerdideoxynucleotide method as the “first generation” approach) have becomewidespread. These newer techniques are characterised by highthroughputs, e.g. as a consequence of the use of parallel, e.g.massively parallel sequencing reactions, or through less time-consumingsteps. Various high throughput sequencing methods provide singlemolecule sequencing and employ techniques such as pyrosequencing,reversible terminator sequencing, cleavable probe sequencing byligation, non-cleavable probe sequencing by ligation, DNA nanoballs, andreal-time single molecule sequencing.

Nucleic acid sequence analysis may also take the form of anoligonucleotide hybridisation probe based approach in which the presenceof a target nucleotide sequence is confirmed by detecting a specifichybridisation event between a probe and its target. In these approachesthe oligonucleotide probe is often provided as part of a wider array,e.g. an immobilised nucleic acid microarray.

Further approaches are available and may be developed in the future, butas a common theme it is typical, albeit not essential, that each areperformed on nucleic acid that has been amplified in a nucleic acidamplification reaction or synthesised by a reverse transcription (RT)reaction. Amplification is typically required to ensure that there issufficient nucleic acid sample for the sequence analysis. Suchtechniques include the polymerase chain reactions (PCRs), ligaseamplification reaction (LAR; also known as ligase chain reaction (LCR)),strand displacement amplification (SDA), nucleic acid sequence basedamplification (NASBA; also known as 3SR (Self-Sustaining SequenceReplication)) and may be preceded by a reverse transcription reaction.These amplification techniques and reverse transcription techniquesoften result in the presence of non-target single stranded DNA in thefinal product, mainly because an excess of single strandedoligodeoxyribonucleotide primer is often supplied initially, but singlestranded DNA amplicons may also arise when polymerisation is incomplete.Such single stranded DNA interfere with the sequence analysis of theamplification product by competing with the other reagents, e.g.oligonucleotide probes, and undergoing sequencing themselves, therebycontaminating the sequencing information outputted by the reaction. Thispotentially lowers the sensitivity and accuracy of the analysis.

To mitigate such interference the product of a nucleic acidamplification reaction or a reverse transcription reaction may betreated with an exonuclease to degrade single stranded DNA (e.g.unincorporated primers). It is also possible to include a treatment toeffect the dephosphorylation of any unincorporated NTPs (e.g. dNTPs),e.g. a treatment with an alkaline phosphatase, for instance theheat-labile shrimp alkaline phosphatase (SAP). Today exonuclease I fromE. coli (ExoI) is the most commonly used exonuclease in such reactions.The exonuclease I reaction is typically performed by adding the enzymeto the product of the nucleic acid amplification or the reversetranscription reaction and incubating for 15 min at 37° C. However, toprevent the exonuclease from interfering with downstream processes, e.g.sequence analysis, it is usually necessary to inactivate the enzyme.With the commonly used exonuclease I from E. coli, inactivation requiresincubation of the enzyme at 80° C. for 15-20 minutes. The longinactivation time and relatively high inactivation temperature makes theprocess time consuming and relatively harsh on the sample of interest.

RecJ and ExoVII have also been proposed for such a use, but are notcommonly used because RecJ has a low specific activity and ExoVIIconsists of two different polypeptide chains. The recommendedinactivation conditions for RecJ involve a 20 min incubation at 65° C.and for ExoVII a 10 minutes incubation at 95° C.

There is a need therefore to provide an enzyme capable of specificallydegrading single stranded DNA in under 15 minutes and/or which may beessentially irreversibly inactivated at a temperature below 65° C.and/or in less than 15-20 minutes. Such a thermolabile (alsointerchangeably referred to herein as a heat-labile (HL)) enzyme wouldimprove molecular biology techniques by making them significantly moretime efficient and gentler on the sample molecule of interest.

It may be desirable to remove single stranded DNA from a sample in othercontexts. For instance, to digest the single stranded DNA to which anucleic acid binding protein is bound. In these contexts an enzymecapable of specifically degrading single stranded DNA in less than 15minutes and/or which may be essentially irreversibly inactivated at atemperature below 65° C. and/or in less than 15-20 minutes would beadvantageous over exonuclease I, RecJ and ExoVII from E. coli for thesame reasons of time efficiency and gentle processing discussed above.

It has now been found that homologs of the E. coli sbcB gene (whichencodes for E. coli exonuclease I) obtained from species of the generaShewanella, Halomonas, Vibrio, Psychromonas and Moritella found in coldwater niches, e.g. Moritella viscosa and Vibrio wodanis surprisinglyhave these advantageous properties.

Therefore, in a first aspect there is provided an exonuclease or anenzymatically active fragment thereof, said exonuclease having the aminoacid sequence of SEQ ID NO:1 or an amino acid sequence which is at leastabout 50% identical thereto, wherein said exonuclease or enzymaticallyactive fragment thereof

(i) is substantially irreversibly inactivated by heating at atemperature of about 55° C. for 10 minutes in a buffer consisting of 10mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl₂;

(ii) is substantially specific for single stranded DNA; and

(iii) has a 3′-5′ exonuclease activity.

By “at least about 50%” it is meant that the sequence identity may be atleast 49%, 49.5% or 49.9%. In preferred embodiments the exonuclease ofthe invention has an amino acid sequence which is at least 60%,preferably at least 70%, 80%, 85%, 90% or 95%, e.g. at least 98% or 99%or 99.5%, identical to SEQ ID NO:1. In other embodiments the exonucleaseconsists of the amino acid sequence of SEQ ID No 1. Enzymatically activefragments thereof are also provided.

An exonuclease having an amino acid sequence which is at least 50%identical to SEQ ID NO:1 may be obtained from a prokaryotic organismfound in cold water niches. By “prokaryote” it is meant any organismthat lacks a cell nucleus, i.e. any organism from the domains Bacteriaand Archaea. Preferably the organism is a bacterium. Preferably theorganism is not a eukaryote, e.g. an organism classified in thetaxonomic kingdoms Animalia, Plantae, Fungi or Protista. More preferablythe organism is selected from the genera Shewanella, Halomonas, Vibrio,Psychromonas and Moritella.

In certain embodiments an exonuclease having an amino acid sequencewhich is at least about 50% identical to SEQ ID NO:1 may be selectedfrom SEQ ID NO:2 (the SbcB homolog from Halomonas sp.), SEQ ID NO:3 (theSbcB homolog from Vibrio wodanis), SEQ ID NO:4 (the SbcB homolog fromPsychromonas sp.) or SEQ ID NO:5 (the SbcB homolog from Moritellaviscosa).

Thus, in another aspect of the invention there is provided anexonuclease or an enzymatically active fragment thereof, saidexonuclease having an amino acid sequence selected from:

-   -   (a) SEQ ID NO:1 or an amino acid sequence which is at least 65%        identical thereto,    -   (b) SEQ ID NO:2 or an amino acid sequence which is at least 65%        identical thereto,    -   (c) SEQ ID NO:3 or an amino acid sequence which is at least 65%        identical thereto,    -   (d) SEQ ID NO:4 or an amino acid sequence which is at least 65%        identical thereto, or    -   (e) SEQ ID NO:5 or an amino acid sequence which is at least 65%        identical thereto,        wherein said exonuclease or enzymatically active fragment        thereof    -   (i) is substantially irreversibly inactivated by heating at a        temperature of about 55° C. for 10 minutes in a buffer        consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5        mM MgCl₂;    -   (ii) is substantially specific for single stranded DNA, and    -   (iii) has a 3′-5′ exonuclease activity.

In preferred embodiments of this aspect of the invention, theexonuclease has an amino acid sequence which is at least 70%, preferablyat least 80%, 85%, 90% or 95%, e.g. at least 98% or 99% or 99.5%,identical to SEQ ID NOs:1, 2, 3, 4 or 5. In other embodiments theexonuclease consists of an amino acid sequence selected from the groupconsisting of SEQ ID NOs:1, 2, 3, 4 and 5. Enzymatically activefragments thereof are also provided.

Percentage sequence identity according to the invention can becalculated using any of the widely available algorithms, e.g. using theClustal W2 multiple sequence alignment program(http://www.ebi.ac.uk/Tools/clustalW2) using default parameters (DNA GapOpen Penalty=15.0; DNA Gap Extension Penalty=6.66; DNA Matrix=Identity;Protein Gap Open Penalty=10.0; Protein Gap Extension Penalty=0.2;Protein matrix=Gonnet; Protein/DNA ENDGAP=−1; Protein/DNA GAPDIST=4)

Variants of the abovementioned SEQ ID NOs include amino acid sequencesin which one or more amino acids of said SEQ ID NOs have undergoneconservative substitution or have been replaced with a modified versionof said one or more amino acids or an amino acid which is not naturallyoccurring, e.g. D isomers of said one or more amino acids. Preferablysuch substitutions and modifications are silent substitutions andmodifications in that the modified forms of the exonucleases of theinvention have the same enzymatic and inactivation characteristics asthe unmodified forms.

An exonuclease is an enzyme capable of cleaving a nucleotide from one ormore termini of a polynucleotide chain, without nucleotide sequencespecificity, by hydrolysing an internucleotide phosphodiester bond.Typically exonucleases cleave nucleotides from either the 5′ terminus(and so are characterised as 5′-3′ exonucleases or as having 5′-3′exonuclease activity) or the 3′ terminus (and so are characterised as3′-5′ exonucleases or as having 3′-5′ exonuclease activity). Inaccordance with the invention the exonucleases are 3′-5′ exonucleases.In some embodiments the exonucleases of the invention have substantiallyno 5′-3′ exonuclease activity against single stranded DNA, by which itis meant that, at concentrations of 0.1 to 1.0 U/μl in a bufferconsisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mMMgCl₂, the exonucleases of the invention display little, negligible oressentially no 5′-3′ exonuclease activity against single stranded DNAover the course of a 1 hour incubation. Expressed numerically, less than10%, e.g. less than 5%, 4%, 3%, 2% or 1%, of a single stranded DNAsubstrate (e.g. about 5 pmol of said substrate, which may for example bean oligodeoxyribonucleotide) under such conditions will be degraded in a5′-3′ direction. Preferably, there will be no detectable 5′-3′exonuclease activity at such concentrations.

The skilled person would be able to devise a suitable assay to measurerelative 5′-3′ and 3′-5′ exonuclease activities. For instance thegel-based exonuclease assay described in the Examples uses a 5′ (FAM)labelled single stranded oligodeoxyribonucleotide to monitor degradationfrom the 3′ terminus as such an activity will yield detectable fragmentsof varying length shortened from the 3′ terminus. Degradation from the5′ terminus will yield only single labelled nucleotides. To confirmthese results the same assay may alternatively be performed with a 3′labelled single stranded DNA substrate and the opposite gel patternshould be observed.

By “substantially specific for single stranded DNA” it is meant that theactivity of the exonuclease of the invention against double stranded DNAis equal to or less than 15% of the activity against an equivalentamount of single stranded DNA under the same conditions, e.g. an enzymeconcentration of about 0.1 U/μl and about 5 pmol of nucleic acidsubstrate in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50mM KCl and 5 mM MgCl₂ with an incubation time of 10 minutes or less andan incubation temperature of about 30° C. In other embodiments theactivity of the exonuclease of the invention against double stranded DNAis equal to or less than 10%, e.g. equal to or less than 8%, 5%, 4%, 3%,2%, 1%, 0.5%, 0.2%, 0.1% or 0.05%, of the activity against an equivalentamount of single stranded DNA under the same conditions.

In further more specific embodiments “substantially specific for singlestranded DNA” also means that the activity of the exonuclease of theinvention against single stranded RNA is equal to or less than 15% ofthe activity against an equivalent amount of single stranded DNA underthe same conditions, e.g. an enzyme concentration of about 0.1 U/μl andabout 5 pmol of nucleic acid substrate in a buffer consisting of 10 mMTris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl₂ with an incubationtime of 10 minutes or less and an incubation temperature of about 30° C.In other embodiments, the activity of the exonuclease of the inventionagainst single stranded RNA is equal to or less than 10%, e.g. equal toless than 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% or 0.05%, of theactivity against an equivalent amount of single stranded DNA under thesame conditions.

In certain embodiments by “substantially specific for single strandedDNA” it is meant that the exonuclease of the invention degrades singlestranded DNA but at concentrations of about 0.1 U/μl in a bufferconsisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mMMgCl₂, there is little, negligible or essentially no detectabledegradation of about 5 pmol of a suitable double stranded DNA substrate(e.g. a double stranded oligodeoxyribonucleotide) over the course of anabout 10 minute incubation. Expressed numerically, equal to or less than15%, e.g. equal to or less than 10%, 8%, 5%, 4%, 3%, 2% or 1%, of thesuitable double stranded DNA substrate will be degraded under suchconditions. Preferably, there will be no detectable degradation ofdouble stranded DNA substrates under such concentrations.

In further more specific embodiments “substantially specific for singlestranded DNA” also means that the exonuclease of the invention degradessingle stranded DNA but at concentrations of about 0.1 U/μl in a bufferconsisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mMMgCl₂, there is little, negligible or essentially no detectabledegradation of about 5 pmol of a suitable single stranded RNA substrate(e.g. a single stranded oligoribonucleotide) over the course of an about10 minute incubation. Expressed numerically, equal to or less than 15%,e.g. equal to or less than 10%, 8%, 5%, 4%, 3%, 2% or 1%, of thesuitable single stranded RNA substrate will be degraded under suchconditions. Preferably, there will be no detectable degradation ofsingle stranded RNA substrates under such concentrations.

In certain embodiments “substantially specific for single stranded DNA”also means that the exonucleases of the invention degrade singlestranded DNA but at concentrations of 0.1 to 1.0 U/μl (preferably 0.1U/μl) in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mMKCl and 5 mM MgCl₂, there is little, negligible or essentially nodetectable degradation of non-single stranded DNA nucleic acidsubstrates over the course of a 1 hour incubation. As used herein, theterm “non-single stranded DNA nucleic acid substrates”, in someembodiments, refers both to double stranded nucleic acids, e.g. doublestranded DNA, and single stranded non-DNA nucleic acid, although inpreferred embodiments the term refers only to double stranded nucleicacids, e.g. double stranded DNA. Expressed numerically, less than 10%,e.g. less than 5%, 4%, 3%, 2% or 1%, of (e.g. about 5 pmol of) asuitable non-single stranded DNA substrate (i.e. double stranded nucleicacid and/or single stranded non-DNA nucleic acid) under such conditionswill be degraded. Preferably, there will be no detectable degradation ofnon-single stranded DNA nucleic acid substrates (i.e. double strandednucleic acid and/or single stranded non-DNA nucleic acid) at suchconcentrations.

The skilled person would easily be able to devise an experiment to makea comparison of relative nuclease activity towards single and doublestranded nucleic acid. For instance, an exonuclease under test may beincubated with two samples of a radioactively labelled PCR product (e.g.about 5 pmol of said PCT product), one in which the product has beendenatured (i.e. single stranded) and the other in which the product isnot denatured (i.e. double stranded) in a buffer consisting of 10 mMTris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl₂ at a concentrationof 0.1 to 1.0 U/μl (preferably 0.1 U/μl) for 10 minutes. The release ofacid soluble nucleotides can then be analysed as described in Examples 8and 9.

Alternatively, an exonuclease under test may be incubated with theabovementioned samples of PCR product (e.g. about 5 pmol of said PCTproduct) in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50mM KCl and 5 mM MgCl₂ at a concentration of 0.1 to 1.0 U/μl (preferably0.1 U/μl) for one hour and then products separated on a suitable,electrophoresis gel e.g. agarose. Activity against single strandedand/or double stranded nucleic acid will be observable by the positionof the bands relative to untreated controls.

Another approach measures the increase in fluorescence fromoligonucleotides labelled with the fluorophore FAM (fluorescein) at the5′ terminus and with TAMRA at the 3′ terminus. The emitted light fromFAM is absorbed (quenched) by TAMRA when the two labels are inproximity. The cleavage of the oligonucleotide by the exonuclease undertest results in the separation of FAM from TAMRA and an increase influorescence from FAM that can be measured in a fluorimeter withexcitation wavelength 485 nm and emission wavelength 520 nm. A doublestranded substrate can be prepared by mixing the labelledoligonucleotide with a second oligonucleotide that is complementary tothe labelled oligonucleotide. Of course other suitable fluorophore pairsmay similarly be used. Example 7 describes a suitable assay in greaterdetail.

Included within the term DNA are modifications thereof which retain aphosphodiester linked deoxyribo-phosphate backbone. Commonly encounteredexamples of single stranded DNA include oligodeoxyribonucleotide primersand oligodeoxyribonucleotide probes and DNA aptamers. Nucleicidentification tags and labels may also be single stranded DNA. Singlestranded DNA also arises during reverse transcription, and upon duplexunwinding during DNA replication and DNA transcription. In nucleic acidamplification reactions and reverse transcription reactions singlestranded DNA may arise from partially extended primers and any excess ofprimers that have not been extended and integrated into completelysynthesised duplexes.

In further embodiments the exonucleases of the invention havesubstantially no endonuclease activity. By “substantially noendonuclease activity” it is meant that, at concentrations of 0.1 to 1.0U/μl in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50 mMKCl and 5 mM MgCl₂, the exonuclease of the invention displays little,negligible or essentially no nuclease activity against a circular singlestranded nucleic acid or circular double stranded nucleic acid over thecourse of a 1 hour incubation. Expressed numerically, less than 10%,e.g. less than 5%, 4%, 3%, 2% or 1%, of (e.g. about 5 pmol of) a singleor double stranded nucleic acid substrate will be fragmented (e.g. intooligonucleotides) under such conditions. Preferably, there will be nodetectable endonuclease activity at such concentrations.

Enzymatically active fragments and variants of SEQ ID Nos. 1-5 displayat least 70%, preferably at least 85%, more preferably at least 95% andmost preferably at least 99% of the enzymatic function of the enzymes ofSEQ ID Nos. 1-5, respectively. As discussed elsewhere, the activity ofan exonuclease can be assessed easily using routine techniques.

In the following discussion, a reference to an exonuclease of theinvention is also a reference to an enzymatically active fragmentthereof, unless context dictates otherwise.

By “substantially irreversibly inactivated” is meant that on heating tothe specified temperature for the specified time and under the specifiedbuffer conditions, the enzyme is at least 90% inactivated, preferably95%, 98%, 99%, 99.5% or 99.9% inactivated. Percentage inactivation canbe conveniently estimated by incubating a suitably labelled (e.g. a 5′FAM labelled) single stranded DNA sample (e.g. a standard PCR primer,for instance a deoxyribonucleic acid primer having the nucleotidesequence of SEQ ID NO:21—GCTAACTACCACCTGATTAC) for 30 minutes eitherwith an inactivated exonuclease or with a non-inactivated exonuclease ina suitable buffer (e.g. Tris, HEPES, PBS) at a suitable pH (e.g. pH 7.5)at a suitable temperature (e.g. 30° C.) and in the presence of Mg²⁺(e.g. 5 mM); separating the reaction products on a suitable gel (e.g.acrylamide/urea gel) by electrophoresis and measuring the relativeintensities of fluorescence of the DNA bands under UV light e.g. asshown in Examples 5 and 6. Alternative methods could be devised by theskilled person to measure relative activities of inactivated andnon-inactivated exonuclease, in particular a buffer consisting of 10 mMTris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl₂ may be used.

Even when the temperature of the reaction mixture returns to roomtemperature, the exonucleases of the invention do not regain activity,i.e. there is substantially no residual activity; specifically, lessthan 10%, preferably less than 5%, 2%, 1%, 0.5% or 0.1%, most preferablyno detectable exonuclease activity remains.

Substantially irreversible inactivation occurs within 10 minutes ofincubation in the specified buffer conditions at a temperature of at orabout 55° C., e.g. 53 to 57° C. For example in 7, 8 or 9 minutesincubation at about 55° C. As shown in Example 5, in other embodimentsthe exonuclease of the invention is substantially irreversiblyinactivated in the specified buffer conditions at a temperature of at orabout 55° C., e.g. 53 to 57° C., within 5 minutes, for example in 2, 3or 4 minutes. In further embodiments the exonuclease of the invention issubstantially irreversibly inactivated in the specified bufferconditions at a temperature of at or about 50° C., e.g. 48 to 53° C.,within 10 minutes, for example in 7, 8 or 9 minutes. In still furtherembodiments the exonuclease of the invention is substantiallyirreversibly inactivated in the specified buffer conditions at atemperature of at or about 50° C., e.g. 48 to 53° C., within 5 minutes,for example in 2, 3 or 4 minutes. The exonucleases represented by SEQ IDNos.1 and 4 and variants thereof are examples of these latterembodiments.

In other embodiments substantially irreversible inactivation of theexonuclease of the invention occurs within 1 minute of incubation in thespecified buffer conditions at a temperature of at or about 80° C., e.g.70 to 90° C., 75 to 85° C., 78 to 82° C. or 79 to 81° C. For example inan incubation of at least about 30, 40, 50 or 55 seconds at about 80° C.

When in use, the exonuclease of the invention may be substantiallyirreversibly inactivated at lower temperatures or over shorter timeperiods depending on the conditions in which the enzyme is being used,e.g. at 55° C. for 5 minutes or at 50° C. for 5 to 15 minutes, e.g. 10to 15 minutes but, in accordance with the invention, heating for 10minutes at about 55° C. in the specified buffer conditions must besufficient to substantially irreversibly inactivate the enzyme. It willbe readily apparent to the skilled person that adjustments to one ofthese two parameters can be compensated for by adjusting the other. Forinstance increasing the inactivation temperature might permit theduration of incubation to be reduced. Conversely, increasing theduration of incubation might permit a lower inactivation temperature tobe used. Of course, as is also readily apparent to the skilled personand shown in the Examples, when the exonucleases of the invention areused in the methods of the invention, durations of incubation longerthan ten minutes may be used and inactivation temperatures greater thanabout 55° C. may be used, if practical (e.g. inactivation could takeplace at 80° C. for 1 minute, 60° C. for 5 to 10 minutes, 55° C. for 15minutes or 50° C. for 15 minutes). However, to be in accordance with theinvention, an exonuclease must show substantial inactivation ifincubated at a temperature of at or about 55° C. for 10 minutes in thespecified buffer conditions.

Inactivation temperatures and times for an exonuclease of the inventionshould be assessed by incubating the exonuclease in buffer consisting of10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl₂. Theexonuclease should be present at about 0.1 to 1.0 U/μl, preferably 0.1to 1.5 U/μl, 0.1 to 5 U/μl or 0.1 U/μl to 10 U/μl.

In most preferred embodiments an exonuclease of the invention may have(or consist of) an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In another aspect of the invention there is provided an exonucleasehaving (or consisting of) an amino acid sequence of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. Being defined entirelyby amino acid sequence means the particular functional featuresdescribed above do not necessarily apply to this aspect. Nevertheless,as shown in the Examples, each of these specific exonucleases findsutility in the methods of the invention described herein.

The exonucleases of the invention may be provided in a modified form,e.g. as a fusion protein with an amino acid motif useful in a processfor the isolation, solubilisation and/or purification or identificationof the exonucleases. Such amino acid motifs (also known as protein tags)include, but are not limited to polyhistidine (His) tags. Examples ofpolyhistidine tagged exonucleases of the invention are recited in SEQ IDNO:11 to 15. Such enzymes form a further aspect of the invention.

Further modifications include the introduction of small chemical groupsto available atoms of the polypeptide, e.g. protecting groups for the Nand C termini or the R-groups of non-essential amino acid residueswithin the polypeptide. In other embodiments the exonucleases of theinvention may be provided immobilised on a solid support, e.g. a solidsupport selected from particles, pellets, beads, sheets, gels, filters,membranes, fibres, capillaries, chips, micro titre strips, slides,tubes, plates or wells etc. Preferably the support is magnetic(preferably paramagnetic or superparamagnetic) e.g. magnetic particles,for instance magnetic beads and pellets. Still further modified formsinclude dimers or trimers of the exonucleases of the invention. Suchentities may be homogeneous or heterogeneous in their monomercomposition.

The invention also provides nucleic acid molecules encoding theexonucleases of the invention and enzymatically fragments thereof.Nucleotide sequences corresponding to the amino acid sequences of SEQ IDNOs:1 to 5 are disclosed in SEQ ID NOs:6 to 10, respectively, and thenucleic acids of the invention may comprise these nucleotide sequences.Nucleotide sequences corresponding to the amino acid sequences of SEQ IDNOs:11 to 15 are disclosed in SEQ ID NOs:16 to 20, respectively, and thenucleic acids of the invention may comprise these nucleotide sequences.Degeneracy of the genetic code means that each of SEQ ID NOs:6 to 10 and16 to 20 are each only one of many possible nucleotide sequencesencoding the amino acid sequences of SEQ ID NOs:1 to 5 and 11 to 15,respectively. Accordingly, the invention extends to nucleic acidmolecules comprising nucleotide sequences which are degenerate versionsof SEQ ID NOs:6 to 10 and 16 to 20. The nucleic acid molecules of theinvention may be nucleic acid vectors, e.g. cloning vectors orexpression vectors. Preferred vectors are plasmids compatible withbacterial and/or yeast cells.

The enzymatic activities and inactivation characteristics of theexonucleases of the invention make such enzymes especially suited forthe removal of single stranded DNA from samples containing biologicalmacromolecules, in particular, the products of a nucleic acidamplification or reverse transcription reactions, e.g. double strandedDNA, RNA and DNA:RNA duplexes.

Thus, in a further aspect there is provided a method of removing singlestranded DNA from a sample, preferably a sample of biologicalmacromolecules, said method comprising contacting the sample with anexonuclease as defined above.

The exonucleases of the invention are thus used to degrade singlestranded DNA present in the sample. In particular, the method involvescontacting the sample with an exonuclease of the invention underconditions which permit the digestion of at least a portion of thesingle stranded DNA present in the sample and then optionally heatingthe sample to inactivate said exonuclease. These steps of digestion andinactivation will typically be incubations and are described herein, inparticular in the Examples. Suitable incubation conditions to achievedigestion of single stranded DNA in a sample are known in the art andmay conveniently comprise incubation at 10 to 45° C., e.g. at or around20 to 40° C. or 30° C. for 1 to 30 minutes, e.g. 1 to 20 minutes, 1 to15 minutes or 1 to 10 minutes, preferably around 5 minutes or less. If atemperature at the higher end of these ranges is used, the duration ofincubation may be at the lower end of these ranges, and vice versa.Inactivation conditions may be based on the discussion of suchparameters given above.

By “removing single stranded DNA” it is meant that the amount of singlestranded DNA in the sample is reduced to some extent. Encompassed areembodiments in which the amount of single stranded DNA is reduced belowdetectable levels, as well as embodiments in which the amount of singlestranded DNA is reduced to a smaller but still detectable amount.Preferably the amount of single stranded DNA is reduced sufficiently toimprove the quality of the sample as defined by the relevant context,e.g. its use in a nucleic acid sequence analysis reaction. Expressednumerically the amount of single stranded DNA in a sample may be reducedby at least 10% e.g. by at least, 20%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95% or 99% or by 100%. In practical terms, to remove singlestranded DNA from a sample, or to reduce the amount of single strandedDNA in a sample, in accordance with the invention, is to degrade atleast a portion of the single stranded DNA present in a sample. The term“at least a portion” should be construed in line with the foregoing.

In a preferred embodiment, the sample is a preparation containing anucleic acid of interest (e.g. DNA, RNA, PNA), e.g. a double strandednucleic acid or a DNA:RNA duplex; a protein of interest, for example arecombinantly produced protein of interest, e.g. an enzyme; acarbohydrate polymer; or a lipid. Alternatively the protein of interestmay be an analyte or other protein which it is desired to purify from astarting material. The protein of interest may be an antibody orantibody fragment. The protein (e.g. antibody) could be useful indiagnostic or therapeutic methods. Thus, the method above described maybe used in order to ensure that the diagnostic or therapeutic protein isfree from contaminating single stranded DNA so that it may be safe toadminister. The protein of interest may be a DNA binding protein orother protein which associates with nucleic acid, particularly singlestranded DNA in solution. Accordingly, the preparation may be derivedfrom a cell lysate or tissue sample or body fluid and/or may be theproduct of a nucleic acid amplification or a reverse transcriptionreaction. The method of the invention can therefore be considered asencompassing a method for refining or enriching a sample comprisingdouble stranded DNA, RNA and/or DNA:RNA duplexes by removing singlestranded DNA. Such a method would comprise contacting the sample with anexonuclease as defined above.

In preferred embodiments the sample is the product of a nucleic acidamplification reaction or the product of a reverse transcriptionreaction and thus the invention provides a method of removing singlestranded DNA from the product of a nucleic acid amplification reactionor the product of a reverse transcription reaction, said methodcomprising the use of an exonuclease as defined above. The method willtypically comprise contacting the product of a nucleic acidamplification reaction or the product of a reverse transcriptionreaction with an exonuclease as defined above.

The exonucleases of the invention are thus used to degrade singlestranded DNA present in the product of a nucleic acid amplificationreaction or the product of a reverse transcription reaction. Inparticular, the method involves contacting the product of a nucleic acidamplification reaction or the product of a reverse transcriptionreaction with an exonuclease of the invention under conditions whichpermit the digestion of at least a portion of the single stranded DNApresent therein and optionally heating the mixture to inactivate saidexonuclease. The features of the steps of digestion and inactivation aredescribed above.

The term “nucleic acid amplification reaction” refers to any in vitromeans for increasing the number of copies of a target sequence ofnucleic acid or its complementary sequence. Preferably, theamplification methods will involve “thermal cycling”, i.e. involvinghigh temperature cycling. Amplification methods include, but are notlimited to, PCR and modifications thereto, 3SR, SDA, LAR or LCR and LAMPand modifications thereto. PCR and LCR and their modifications arethermal cycling methods. Methods may result in a linear or exponentialincrease in the number of copies of the target sequence. “Modifications”encompass, but are not limited to, real-time amplification, quantitativeand semi-quantitative amplification, competitive amplification, hotstart PCR, and so on. Reverse transcription maybe combined with othernucleic acid amplification reactions as appropriate.

Preferably the nucleic acid amplification method is a method based onthe use of oligonucleotide primers as initiators of nucleic acidsynthesis, e.g. the PCRs, LAR, SDA, LAMP and NASBA.

The target nucleic acid for the amplification reaction may be DNA or RNAdepending on the selected amplification method. For example, for PCR thetarget is DNA, although when combined with a reverse transcription stepthe target can be considered to be an RNA sequence. 3SR amplifies RNAtarget sequences directly.

“Reverse transcription” is a process in which a single strand RNAtemplate is transcribed into a complementary single stranded DNA (cDNA).These nucleic acid strands exist as a duplex until denaturing conditionsare applied. The single stranded DNA may also then be used as a templateto form double stranded cDNA in a so-called second strand cDNA synthesisstep. Some nucleic acid polymerase enzymes are capable of producing thefirst cDNA strand and synthesising the second strand to form doublestranded cDNA and others are specific for just one of the two steps.

A “product of a nucleic acid amplification reaction” is thereforeconsidered to comprise essentially all of the components obtaineddirectly from the final amplification step of the reaction in question.Other components may be added or certain of the components may undergosome modification or processing, but essentially none of the components,or at least none of the nucleic acid components, will have been removed.Preferably the product of a nucleic acid amplification reaction is thedirect product of the final amplification step; however, it might alsobe preferable for the product of a nucleic acid amplification reactionto undergo a treatment to effect the dephosphorylation of anyunincorporated NTPs, e.g. a treatment with an alkaline phosphatase,preferably a thermolabile alkaline phosphatase, for instance theheat-labile shrimp alkaline phosphatase (SAP), prior to treatment withthe exonucleases of the invention. References to NTPs hereinspecifically include references to dNTPs unless context dictatesotherwise. In other embodiments the dephosphorylation of NTPs may takeplace after treatment with the exonucleases of the invention, or at thesame time. An advantageous recombinant SAP is available fromArcticZymes™ AS.

Accordingly, the above defined methods of removing single stranded DNAfrom the product of a nucleic acid amplification reaction or the productof a reverse transcription reaction may further comprise using analkaline phosphatase, preferably a thermolabile alkaline phosphatase, todephosphorylate any unincorporated NTPs prior to, at the same time as,or after using the exonuclease to remove single stranded DNA, i.e. priorto, at the same time as, or after the step of contacting the product ofthe amplification or reverse transcription reaction with theexonuclease. In particular, using an alkaline phosphatase in thesemethods involves contacting the alkaline phosphatase with the product ofthe amplification/reverse transcription reaction prior to, at the sametime as, or after contacting said product with the exonuclease of theinvention.

Preferably, any alkaline phosphatase treatment may precede any heatinactivation step. In embodiments where it does not, a further heatinactivation step may be used.

In structural terms, a product of a nucleic acid amplification reactionin accordance with the invention will comprise the template nucleic acidand NTPs, and typically in addition a polymerase and at least oneoligonucleotide primer, which may be partially extended. Also typicallythe bulk of the product will be made up of a suitable nucleic acidamplification buffer, e.g. a buffer as exemplified herein. Preferredproducts of nucleic acid amplification reactions comprise all of theseelements.

In these embodiments the treatments of the invention remove (i.e.degrade), as appropriate, excess non-extended DNA primers, partiallyextended DNA primers, single stranded DNA template, single stranded DNAamplicons, denatured DNA amplicons and so on.

A “product of a reverse transcription reaction” should be construed inaccordance with the definition of a product of a nucleic acidamplification reaction, keeping in mind that a reverse transcriptionreaction might have only one RNA-dependent nucleic acid polymerisationstep and, optionally, may have one or more second strand cDNA synthesisstep(s). Preferably the product of a reverse transcription reaction towhich the exonuclease of the invention is applied is a productcomprising cDNA:RNA duplexes and/or double stranded cDNA.

In a further aspect of the invention there is provided a method ofnucleic acid amplification, said method comprising a step subsequent tothe final amplification step of using an exonuclease as defined hereinto remove single stranded DNA from the product of the nucleic acidamplification reaction. Typically, the step of removing single strandedDNA comprises contacting the product of the nucleic acid amplificationreaction, preferably the direct product of the final amplification step,with an exonuclease as defined herein. Said method may further comprisea step, subsequent to the final amplification step, of using an alkalinephosphatase, preferably a thermolabile alkaline phosphatase, todephosphorylate any unincorporated NTPs in the product of the nucleicacid amplification reaction prior to, at the same time as, or afterusing the exonuclease to remove single stranded DNA, i.e. prior to, atthe same time as, or after the step of contacting the product of theamplification reaction with the exonuclease. In particular, using analkaline phosphatase in these methods involves contacting the alkalinephosphatase with the product of the amplification reaction prior to, atthe same time as, or after contacting said product with the exonucleaseof the invention.

In a further aspect of the invention there is provided a method ofreverse transcription, said method comprising a step subsequent to thefinal reverse transcription step, and/or, if present, the final secondstrand cDNA synthesis step, of using an exonuclease as defined herein toremove single stranded DNA from the product of the reverse transcriptionreaction. Typically, the step of removing single stranded DNA comprisescontacting the product of the reverse transcription reaction, preferablythe direct product of the final reverse transcription step, and/or, ifpresent, the direct product of the final second strand cDNA synthesisstep, with an exonuclease as defined herein. Preferably such productswill be products comprising cDNA:RNA duplexes and/or double strandedcDNA. Said method may further comprise a step, subsequent to the finalreverse transcription step, and/or, if present, the final second strandcDNA synthesis step, of using an alkaline phosphatase, preferably athermolabile alkaline phosphatase, to dephosphorylate any unincorporatedNTPs in the product of the reverse transcription reaction prior to, atthe same time as, or after using the exonuclease to remove singlestranded DNA, i.e. prior to, at the same time as, or after the step ofcontacting the product of the reverse transcription reaction with theexonuclease. In particular, using an alkaline phosphatase in thesemethods involves contacting the alkaline phosphatase with the product ofthe reverse transcription reaction prior to, at the same time as, orafter contacting said product with the exonuclease of the invention.

The exonucleases of the invention are thus used to degrade singlestranded DNA present in the product of a nucleic acid amplificationreaction or the product of a reverse transcription reaction. Inparticular, the methods involve contacting the product of a nucleic acidamplification reaction or the product of a reverse transcriptionreaction with an exonuclease of the invention under conditions whichpermit the digestion of at least a portion of the single stranded DNApresent therein and optionally heating the mixture to inactivate saidexonuclease. The features of the steps of digestion and inactivation aredescribed above. Preferably, any alkaline phosphatase treatment mayprecede the heat inactivation step. In embodiments where it does not, afurther heat inactivation step may be used.

It is commonplace to combine reverse transcription with one or morenucleic acid amplification reactions. It is also commonplace to combinea plurality of nucleic acid amplification reactions into a singlemultistage protocol. It will be immediately apparent that each part ofthese multistage protocols may be considered a method of nucleic acidamplification or method of reverse transcription of the invention intheir own right and thus the exonuclease of the invention may be used inaccordance with the invention in any or all parts of such multistageprotocols.

In a further aspect of the invention there is provided a method ofoptimising nucleic acid sequence analysis, said method comprising usingan exonuclease as defined herein to remove single stranded DNA from thesample to be analysed. The method will typically comprise contacting thesample with an exonuclease as defined herein.

Preferably in this aspect the sample is the product of an amplificationreaction or the product of a reverse transcription reaction, e.g. asdefined above, but other samples may be used, e.g. those prepareddirectly from biological materials or which contain biologicalmaterials, e.g. microorganisms, body fluids, eukaryotic cells, cultures,tumours and tissues. Preferably the sample will be at least partiallypurified nucleic acid preparations of the aforementioned samples, e.g.DNA and RNA preparations.

The term “optimisation” encompasses an improvement in the accuracyand/or sensitivity of the sequence analysis of nucleic acids (e.g. theproducts of nucleic acid amplification or reverse transcriptionreactions). In accordance with the invention this improvement is, atleast in part, a result of the removal of single stranded DNA which mayinterfere and/or compete with the sequence analysis reactions, reducingtheir effectiveness against the target template, and/or which arethemselves sequenced and so contribute to the background noise in thesystem, thus making analysis of the outputted signals less sensitive.

In a further aspect of the invention there is provided a method ofnucleic acid sequence analysis, said method comprising a step of samplepreparation prior to the analysis step(s) of using an exonuclease asdefined herein to remove single stranded DNA from the sample to beanalysed. The method will typically comprise contacting the sample withan exonuclease as defined herein.

In these latter aspects where the sample is a product of anamplification reaction or the product of a reverse transcriptionreaction, the sample may also undergo treatment with an alkalinephosphatase, preferably a thermolabile alkaline phosphatase, todephosphorylate any unincorporated NTPs. Accordingly, the above definedmethod of nucleic acid sequence analysis may comprise a further samplepreparation step of using an alkaline phosphatase to dephosphorylate anyincorporated NTPs prior to, at the same time as, or after using theexonuclease to remove single stranded DNA, i.e. prior to, at the sametime as, or after the step of contacting the sample to be analysed withthe exonuclease. In particular, using an alkaline phosphatase in thesemethods involves contacting the alkaline phosphatase with the sample tobe analysed prior to, at the same time as, or after contacting saidproduct with the exonuclease of the invention.

In these latter aspects the exonucleases of the invention are thus usedto degrade single stranded DNA present in the sample to be analysed,e.g. those samples discussed above. In particular, the method involvescontacting the sample with an exonuclease of the invention underconditions which permit the digestion of at least a portion of thesingle stranded DNA present therein and optionally heating the mixtureto inactivate said exonuclease. The features of the steps of digestionand inactivation are described above. Preferably, any alkalinephosphatase treatment may precede the heat inactivation step. Inembodiments where it does not, a further heat inactivation step may beused.

Preferably in these latter aspects the nucleic acid sequence analysis isa sequencing technique, e.g. the Sanger dideoxynucleotide sequencingmethod or a “next generation” or “second generation” sequencing approach(for instance, those involving pyrosequencing, reversible terminatorsequencing, cleavable probe sequencing by ligation, non-cleavable probesequencing by ligation, DNA nanoballs, and real-time single moleculesequencing) or an oligonucleotide hybridisation probe based approach inwhich the presence of a target nucleotide sequence is confirmed bydetecting a specific hybridisation event between a probe and its target.

The nucleic acid sequence analysis may provide information useful in thegenotyping of an organism, e.g. for classification, identification,quantification, prognostic, diagnostic and/or forensic applications, oruseful in the profiling of the transcriptome of a cell or group ofcells, e.g. for prognostic, diagnostic and/or research applications. Theinvention encompasses the use of the exonucleases defined herein in themethods described here for such purposes.

The invention also provides the use of an exonuclease as defined hereinto remove single stranded DNA from a sample, and more specifically inthe methods described herein.

Where appropriate, the exonucleases of the invention may be isolatedfrom a natural source, e.g. isolated from extracts of the organismsdescribed above, or produced recombinantly in a host cell and isolatedand purified therefrom. The exonucleases of the invention may thereforebe recombinant enzymes, in particular isolated recombinant enzymes. Incertain embodiments the exonuclease is produced by recombinanttechniques in a host cell that is not, or not from, an organism which isthe same as that in which the exonuclease is found naturally, i.e. aheterologous host cell. Alternatively, a cell-free expression system canbe used for production of the exonuclease. These approaches may resultin an altered glycosylation pattern.

A method for the isolation and purification of an exonuclease or anenzymatically active fragment thereof as described herein represents afurther aspect of the present invention. Thus, in this aspect theinvention provides such a method, said method comprising culturing cellsin which the exonuclease is expressed and subsequently separating theexonuclease from said cells and/or the media in which said cells havebeen cultured. Preferably the method comprises expressing saidexonuclease in a suitable heterologous host cell (e.g. E. coli, Bacillusand Pichia pastoris), and subsequently separating the exonuclease fromsaid host cells and/or the media in which said cells have been cultured.Expression of said exonuclease can be achieved by incorporating into asuitable host cell an expression vector encoding said nuclease, e.g. anexpression vector comprising a nucleic acid molecule encoding any of theamino acid sequences of SEQ ID NOs. 1 to 5 or 11 to 15, for instance anucleic acid molecule comprising the nucleotide sequence of any of SEQID NOs. 6 to 10 or 16 to 20. Host cells comprising these expressionvectors and nucleic acid molecules are encompassed by the invention.

The exonuclease enzyme may be separated, or isolated, from the hostcells/culture media using any of the purification techniques for proteinknown in the art and widely described in the literature or anycombination thereof. Such techniques may include for example,precipitation, ultrafiltration, dialysis, various chromatographictechniques, e.g. gel filtration, ion-exchange chromatography, affinitychromatography, electrophoresis, centrifugation etc. As discussed above,the exonuclease of the invention may be modified to carry amino acidmotifs or other protein or non-protein tags, e.g. polyhistidine tags, toassist in isolation, solubilisation and/or purification oridentification. Examples of polyhistidine tagged exonucleases of theinvention are recited in SEQ ID Nos 11 to 15.

Likewise an extract of host cells may also be prepared using techniqueswell known in the art, e.g. homogenisation, freeze-thawing etc. and fromthis extract the exonuclease of the invention can be purified.

Freezer storage of the exonuclease of the invention may be convenientlyachieved with a storage buffer of 5 mM Tris/HCl, pH 7.5 (at 25° C.), 250mM NaCl, 5 mM MgCl₂, 0.25 mM EDTA, 50% glycerol, although other buffersmay be used.

The present invention also provides kits which comprise at least anexonuclease according to the invention or a nucleic acid encoding anexonuclease according to the invention. The kits may also contain someor all of the necessary reagents, buffers, enzymes etc. to carry outnucleic acid amplification and/or reverse transcription and/or sequenceanalysis reactions. More particularly, the kits may contain nucleotidetriphosphates (including dNTPaS for SDA), oligonucleotide primers,oligonucleotide probes, reverse transcriptases, DNA polymerases,preferably a thermostable polymerase such as Taq polymerase or Bstpolymerase (and hot-start versions thereof) or, in the case of LAR, aDNA ligase (preferably a thermostable DNA ligase such as Ampligase® orthat disclosed in U.S. Pat. No. 6,280,998 which is isolated fromPyrococcus furiosus) an alkaline phosphatase, preferably a thermolabilealkaline phosphatase (e.g. SAP), or a restriction enzyme (preferably athermostable restriction enzyme such as BsoB1). Kits comprising anexonuclease of the invention and an alkaline phosphatase, preferably athermolabile alkaline phosphatase (e.g. SAP), e.g. any of thosedisclosed herein are of note.

The present invention also provides compositions comprising anexonuclease of the invention and one or more of the necessary reagentsto carry out nucleic acid amplification and/or reverse transcriptionand/or sequence analysis reactions and methods, e.g. those componentsdescribed above. Typically such compositions will be aqueous andbuffered with a standard buffer such as Tris, HEPES, etc. The presentinvention also provides compositions comprising an exonuclease of theinvention in a buffer.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described by way of non-limiting Examples withreference to the following figures in which:

FIG. 1 shows the amino acid and nucleotide sequences of Shewanella sp.exonuclease (SEQ ID No 1 and SEQ ID NO:6, respectively).

FIG. 2 shows the amino acid and nucleotide sequences of Halomonas sp.exonuclease (SEQ ID No 2 and SEQ ID NO:7, respectively).

FIG. 3 shows the amino acid and nucleotide sequences of Vibrio wodanisexonuclease (SEQ ID No 3 and SEQ ID NO:8, respectively).

FIG. 4 shows the amino acid and nucleotide sequences of Psychromonas sp.exonuclease (SEQ ID No 4 and SEQ ID NO:9, respectively).

FIG. 5 shows the amino acid and nucleotide sequences of Moritellaviscosa (SEQ ID NO:5 and SEQ ID NO:10, respectively).

FIG. 6 shows an alignment of SEQ ID Nos:1-5 with the amino acid sequenceof E. coli exonuclease I (SEQ ID NO:22) generated with ClustalW Multiplealignment tool. The consensus sequence is shown at the bottom. *,identical residues in all sequences; highly conserved residues among thesequences; weakly conserved residues among the sequences.

FIG. 7 shows the amino acid and nucleotide sequences of a His-taggedversion of Shewanella exonuclease (SEQ ID No 11 and SEQ ID NO:16,respectively).

FIG. 8 shows the amino acid and nucleotide sequences of a His-taggedversion of Halomonas exonuclease (SEQ ID NO:12 and SEQ ID No. 17,respectively).

FIG. 9 shows the amino acid and nucleotide sequences of a His-taggedversion of Vibrio wodanis exonuclease (SEQ ID NO:13 and SEQ ID NO:18,respectively).

FIG. 10 shows the amino acid and nucleotide sequences of a His-taggedversion of Psychromonas exonuclease (SEQ ID NO:14 and SEQ ID NO:19,respectively).

FIG. 11 shows the amino acid and nucleotide sequences of a His-taggedversion of Moritella viscosa exonuclease (SEQ ID NO:15 and SEQ ID NO:20,respectively).

FIGS. 12A-12D shows images of a number of polyacrylamide gels on whichthe products of a variety of reactions between a single stranded DNAoligonucleotide and a variety of heat-labile (HL) exonucleases have beenseparated, thus indicating activity of the enzyme against singlestranded DNA. Buffer conditions as described in Example 2. (−)Cont—negative control. FIG. 12A: HL-ExoI (Ha) activity at differenttemperatures (20 to 37° C.) at different time intervals (1 to 10minutes). Different dilution factors depending on incubation time (4×for 1 to 5 minutes and 10× for 10 minutes). FIG. 12B: HL-ExoI (Ps)activity at different temperatures (20 to 37° C.) at different timeintervals (1 to 10 minutes). Different dilution factors depending onincubation time (3× for 1 minute, 8× for 5 minutes and 12× for 10minutes). FIG. 12C: HL-ExoI (Sh) activity at different temperatures (20to 37° C.) at different time intervals (1 to 10 minutes). Differentdilution (4× for 1 minute and 5 minutes and 10× for 10 minutes). FIG.12D: HL-ExoI (Mv) activity at different temperatures (20 to 37° C.) atdifferent time intervals (1 to 10 minutes). Different dilution factorsdepending on incubation time (1× for 1 minute and 5 minutes and 2× for10 minutes).

FIG. 13 shows an image of a polyacrylamide gel on which the products ofa variety of reactions between a single stranded DNA oligonucleotide anda variety of heat treated exonucleases have been separated, thusindicating activity of the enzymes against single stranded DNA. Bufferconditions as described in Example 3. (−) Cont— negative control. Fourcommercially available E. coli ExoI enzymes (ExoI A-D), one commerciallyavailable enzymatic PCR clean-up kit and HL-ExoI (Sh) were compared interms of ease of thermal inactivation. Samples were incubated for 1 minat 80° C. before substrate addition and residual activity incubation.

FIGS. 14A-14C shows representative sequencing results from thesequencing reactions of Example 4. FIG. 14A: reaction based on GoTaq PCRbuffer—ExoSAP-IT corresponds the 30 minutes protocol described inExample 4 and HL-ExoI (Sh)/SAP corresponds to the 5 minutes protocoldescribed in Example 4. FIG. 14B: as FIG. 14A, respectively, althoughTEMPase Extra PCR buffer used in place of GoTaq. FIG. 14C: as FIG. 14Aalthough ExoSAP-IT and HL-ExoI (Sh)/SAP corresponds to the 5 minprotocol described in Example 4.

FIGS. 15A-15E shows images of a number of polyacrylamide gels on whichthe products of a variety of reactions between single stranded DNAoligonucleotides and HL-ExoI of the invention have been separated, thusindicating the residual activity of the enzyme following heat treatmentagainst single stranded DNA. Buffer conditions as descried in Example 5.15A: HL-ExoI (Ha); 15B: HL-ExoI (Sh); 15C: HL-ExoI (Ps); 15D: HL-ExoI(Mv); 15E: HL-ExoI (Vw). (−) Cont—negative control. Samples wereincubated for 5 minutes, 10 minutes or 15 minutes at differenttemperatures (40° C.-60° C.) prior to substrate addition and residualactivity incubation, which was performed at 30° C. for 30 minutes, andthen for 2 minutes at 95° C.

FIGS. 16A-16E shows images of a number of polyacrylamide gels on whichthe products of a variety of reactions between single stranded DNAoligonucleotides and HL-ExoI of the invention have been separated, thusindicating the activities of the test ExoI against single stranded DNAat increasing dilution. Buffer conditions as descried in Example 6. 16A:HL-ExoI (Ha); 16B: HL-ExoI (Sh); 16C: HL-ExoI (Ps); 16D: HL-ExoI (Mv);16E: HL-ExoI (Vw). (−) Cont—negative control. 100%—undiluted enzyme, 10%—enzyme diluted 10 times, 1%—enzyme diluted 100 times, 0.1%—enzymediluted 1000 times, 0.01%—enzyme diluted 10,000 times. Samples wereincubated for 30 minutes at 30° C., and then for 2 minutes at 95° C.

FIG. 17 shows the 3′ to 5′ directionality of HL-ExoI (Ha, Ps, Sh, Vw,Mv) as well as for E. coli ExoI. Assay conditions are described inExample 11. When FAM was labeled at the 5′ end, a ladder of partialfaint and intense intermediate product bands were seen indicating theExoI degrading the substrate from the 3′ end. When the oligo wasFAM-labeled at the 3′ end, the fluorophore was immediately cut off,generating only the 3′FAM monomer. (−)Cont—negative control. ExoI—E.coli ExoI.

FIG. 18 shows the crystal structure of HL-ExoI (Mv) in complex withssDNA (dT13) at a resolution of 2.5 Å. Experimental set-up is describedin Example 12. Active site residues Asp23, Glu25 and Asp194 areindicated as sticks. In the three-dimensional structure of the HL-ExoI(Mv) the 3′-end of the dT13 is clearly located in the active site of theenzyme.

FIG. 19 shows a polyacrylamide gel on which the activity andinactivation of HL-ExoI (Ps, Sh, Vw, Mv) and E. coli ExoI were compared.Buffer conditions and reaction set up are as described in Example 12.All enzymes were tested for activity at 30° C. for 15 minutes as well asresidual activity under the same time and temperature conditionsfollowing incubation at 80° C. for 1 minute. To mimic a post-PCRclean-up assay, reaction was performed in a post-PCR buffer.

FIG. 20 shows a polyacrylamide gel on which the activity andinactivation of HL-ExoI (Sh) and E. coli ExoI were compared. Bufferconditions and reaction set up are as described in Example 12. Allenzymes were incubated at 80° C. for 1, 5, 10 or 20 minutes beforesubstrate addition and incubation at 30° C. for 15 minutes. To mimic apost-PCR clean-up assay, reaction was performed in a post-PCR buffer. Noresidual activity in HL-ExoI (Sh) was detected after 1 min incubation at80° C., while substantial residual activity was observed with twocommercial E. coli ExoI after 5 minutes, and even after 20 minutesincubation at 80° C. in the case of ExoI A.

FIGS. 21A-21F shows representative sequencing results from thesequencing reactions of Example 14 (A: Negative control; B: ExoSAP-IT;C: HL-ExoI (Sh)/SAP; D: HL-ExoI (Ps)/SAP; E: HL-ExoI (Mw)/SAP; F:HL-ExoI (Vw)/SAP. All images show the resulting chromatograms followingaddition of excess reverse primer prior to PCR clean-up. All sequencingresults were based upon the GoTaq PCR buffer. ExoSAP-IT corresponds tothe 30 minutes protocol, while HL-ExoI/SAP corresponds to the 5 minutesprotocol, both described in Example 14.

EXAMPLES Example 1—Cloning, Recombinant Expression and PartialPurification of Exonucleases

The sbcB exodeoxyribonuclease I (exoI) gene from Moritella viscosa(HL-ExoI (Mv)), Vibrio wodanis (HL-ExoI (Vw)), Halomonas (HL-ExoI (Ha)),Psychromonas (HL-ExoI (Ps)) and Shewanella (HL-ExoI (Sh)) gDNA wascloned by overlap extension PCR cloning (Bryksin and Matsumura, 2010,Biotechniques, 48 (6), 463-465) with a C-terminal His-tag by insertingthe cloned gene into the pTrc99A expression vector for expression in E.coli TOP10. The primers used are listed in Table 1. Genetic sourcematerial was obtained from bacteria isolated from Norwegian offshorewaters.

TABLE 1 Primers used for cloning of ExoI genes. Primer SEQ ID nameSequence No Mv forward GTGAGCGGATAACAATTTCACACAGGAAACAGACCATGG 23ATAACAATTCGAACAAAACAGCAACAG Mv reverseGCTGAAAATCTTCTCTCATCCGCCAAAACAGCCtcagtgatggt 24gatggtgatg-gcctgcagaTGCGCCAATTATTTTTTGACCATAAAGG Vw forwardGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCC 25 GCAGGATAACGCACCAAGVw reverse GCTGAAAATCTTCTCTCATCCGCCAAAACAGCCtcagtgatggtga 26tggtgatggcctgcagaTGATACTAACTGTTGTACGTAATTATAAACG GCGC Ha forwardGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCA 27 TCACCCAATGCTGCC Ha reverseGCTGAAAATCTTCTCTCATCCGCCAAAACAGCCtcagtgatggtga 28tggtgatggcctgcagaGGCATCAAATGCCTGGGCCG Ps forwardGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAT 29 CAAGAATCCCCAAGCCTTCTTTGGPs reverse GCTGAAAATCTTCTCTCATCCGCCAAAACAGCCtcagtgatggtga 30tggtgatggcctgcagaTGTATTCCCTGTCAAAAACTCTAAGTAATGT CC Sh forwardGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAC 31AACACTAAGAAACAGCCAACTTTATTTTGG Sh reverseGCTGAAAATCTTCTCTCATCCGCCAAAACAGCCtcagtgatggtga 32tggtgatggcctgcagaAAGATTTCTAAGATAATGACACAAAGCCTGT AA Bold letters,pTrc99A specific sequence; upper case letters, gene specific sequence;lower case letters, spacer and tag sequence.

The pTrc99A-exoI vectors were transformed into E. coli TOP10 followingthe protocol for Z-competent cells (Zymo Research, U.S.A.). The cellswere grown in baffled shake flasks in Terrific Broth (TB) medium;approximately 1.5% overnight precultures were transferred to 250 ml TBmedium containing 100 μg/ml Ampicillin in 1000 ml growth flasks andincubated at 37° C., 200 rpm, until the OD₆₀₀ reached 0.4-0.6. Thetemperature was decreased to 15° C. and the cells were incubated for 30minutes before they were induced for 4 hours with 0.5 mM IPTG. The cellswere harvested by centrifugation and the cell pellets frozen at −20° C.

Cell pellets from the 250 ml cultures were thawed on ice, 40 ml lysisbuffer (50 mM Tris-HCl (pH 7.5 at 25° C.), 5 mM imidazole, 1 M NaCl,0.1% Triton X-100, 10% glycerol, 10 mM MgCl₂) was added and the mixturewas sonicated in an ice water bath for 10 min (25% amplitude, 0.1 secon, 0.2 sec off) using a Branson Sonifier. The lysate was centrifuged ina 50 ml tube at 25,000 g for 20 minutes and the supernatant filteredthrough a 0.45 μM filter. The filtered lysate was diluted with 50 ml oflysis buffer to a total volume of 90 ml. All purification steps wereperformed with ice cold buffers and a column cooled on ice. The 90 mllysate was applied to a HisTrap HP 1 ml column equilibrated in lysisbuffer using a flow of 1 ml/min. The column was washed with 5 columnvolumes (CV) of lysis buffer and 10 CV of buffer A2 (50 mM Tris-HCl, (pH7.5 at 25° C.), 5 mM imidazole, 500 mM NaCl). The protein was theneluted with 20 CV of a 0-30% gradient of buffer B (50 mM Tris-HCl, (pH7.5 at 25° C.), 500 mM Imidazole, 500 mM NaCl) to buffer A2 in 1 mlfractions. Fractions containing ExoI activity were pooled and eitherdialysed against 10 mM Tris-HCl (pH 7.5 at 25° C.), 500 mM NaCl and 0.5mM EDTA, or 10 mM Tris-HCl (pH 7.5 at 25° C.), 500 mM NaCl, 10 mM MgCl₂and 0.5 mM EDTA, and then diluted 1:1 with 100% glycerol and stored at−20° C.

Two more exonuclease I sequences (not disclosed) have been examined, butfailed to express and isolate actively.

Example 2—Activity Profiling of Exonucleases: Optimum Temperature forCatalytic Activity

A temperature profile was created to better characterise the differentrecombinant HL-ExoI from Example 1. The different HL-ExoI were dilutedto concentrations enabling differentiation between substratedegradations on the gel. Due to the different dilution factors, samplescould not directly be compared to each other, but relativetemperature-dependent differences in activity could be estimated.Samples were incubated for various time intervals to determine if theenzyme could sustain a set temperature for longer periods of time.

Detailed Method

HL-ExoI was diluted (10 mM Tris-HCl pH 7.5 at 25° C., 5 mM MgCl₂) towhat was thought to give the best differentiation between samples. Tomimic the PCR clean-up protocol, post-PCR solutions were used asreaction buffers. Robustness was achieved by running the experiment inparallel using post-PCR solutions based on two different PCR buffers;GoTaq (Promega) or TEMPase Key (VWR). As substrate, 5 pmol of a 5′ FAMlabeled oligonucleotide (GCTAACTACCACCTGATTAC; SEQ ID No 21) was addedto each reaction. Following addition of the ExoI, the total volume foreach sample was 7 μl. Samples were incubated in a thermocycler for 1minute, 5 minutes or 10 minutes at 20° C., 25° C., 30° C. or 37° C.followed by 5 minutes at 80° C. A TBE-Urea Sample Buffer (Bio-Rad) wasadded and samples were applied to a casted 20% Acrylamide/7M Urea geland run at 180 V for approximately 45 minutes. All reagents were kept onice during the full protocol unless specified otherwise and workflow wasperformed on cooling blocks.

Results

Results are shown in FIG. 12. In general, all HL-ExoI performedsimilarly in the two buffers used to prepare the post-PCR samples,showing that the observed effects were not specific to the PCR-buffercomposition.

HL-ExoI (Ha) showed an overall increasing activity up to 37° C. andcould withstand this temperature for at least 10 minutes (FIG. 12A).Good overall activity at lower temperatures was also seen.

HL-ExoI (Ps) showed an overall best activity at 30° C., with loss ofactivity when using 37° C. as incubation temperature (FIG. 12B). Goodoverall activity at lower temperatures was also seen.

HL-ExoI (Sh) showed an overall best activity at 37° C., and couldwithstand this temperature for at least 10 minutes (FIG. 12C). Goodoverall activity at all temperatures was also seen.

HL-ExoI (Mv) showed an overall best activity at 30° C., while incubationat 37° C. resulted in loss of activity (FIG. 12D). Good activity atlower temperatures was also seen.

Using PAGE for activity profiling appeared to give good estimates as tohow the HL ExoI behaved depending on temperature over time.

Example 3—Activity Profiling of Exonucleases: Inactivation Temperaturesfor Catalytic Activity

In this Example, the thermal inactivation characteristics of HL-ExoI(Sh) were compared to various commercially available E. coli ExoI.

The thermal inactivation characteristics of four different commerciallyavailable E. coli ExoI and one commercially available enzymatic PCRclean-up kit were compared to HL-ExoI (Sh). To ensure that any observedeffects were irrespective of the choice of reaction buffer, twodifferent post-PCR solutions were used as a reaction buffer (TEMPaseKey, TEMPase Extra, VWR). All reactions received about 10 U of ExoI toenable comparison between the ExoI activities and ease of thermalinactivation. The exonuclease activity of HL-ExoI (Sh) was calculated asdescribed in Example 8. For the commercial ExoIs, exonuclease activitywas taken as that stated by the manufacturers. Final volume for eachreaction was 7 μl. Samples were incubated at 80° C. for 1 minute beforecooling and addition of 5 pmol of a 5′ FAM labeled oligonucleotide(GCTAACTACCACCTGATTAC; SEQ ID No 21). Samples were further incubated at37° C. for 15 minutes. Following incubation, a TBE-Urea Sample Buffer(Bio-Rad) was added and samples were applied to a casted 20%acrylamide/7 M urea gel and run at 180 V for approx. 45 minutes. Allreagents were kept on ice during the full protocol and workflow wasperformed on cooling blocks unless specified otherwise.

Results are shown in FIG. 13. All of the E. coli ExoI had adequateactivity following 1 minute incubation at 80° C. to completely degradeall of the substrate. HL-ExoI (Sh) was the only ExoI that was completelyinactivated, showing no signs of residual activity.

Example 4—Demonstration of Utility of HL-ExoI (Sh) in a Rapid PCRClean-Up Prior to Nucleic Acid Sequencing

Experiments were set up to verify that HL-ExoI (Sh) could performsatisfactorily in a rapid PCR clean-up scenario. For comparison andpositive control, parallel samples were treated with a leading brand ofenzymatic PCR clean-up reagent ExoSAP-IT (Affymetrix).

To verify that HL-ExoI (Sh) enabled a 5 minute enzymatic PCR clean-upprotocol, an experiment was designed to stress-test the protocollimitations. Thus, to all post-PCR solutions under test there were addedexcess primers or dNTPs following the PCR. If left unremoved prior tothe sequencing reaction, residual primers would result in sequencereactions in the opposite direction and thereby strongly compromise thelength and quality of the reaction, and dNTPs would result in ddNTP:dNTPratios which would fail to yield high quality sequences. Robustness wasachieved by using different PCR reagents and amplicons.

Following PCR, 10 pmol primers or 40 nmol dNTPs were added to thepost-PCR solutions.

Samples to be treated with ExoSAP-IT were handled according tomanufacturer protocol. Following addition of either reverse primer ordNTP to the PCR solution, samples received 2 μl of the clean-up reagent,giving a final volume of 7 μl. Samples were incubated 15 minutes at 37°C. followed by 15 minutes at 80° C. Samples were set up using twodifferent PCR buffers and all samples were set up as triplicates.

Samples to be treated with HL-ExoI (Sh) received the same amount ofadded primers and dNTPs as above, before addition of 1 μl of HL-ExoI(Sh) (10 U/μl, as calculated in Example 8) and 1 μl of SAP (2 U/μl). Aswith the positive controls, the final volume of the samples were 7 μl.Samples were incubated 4 minutes at 37° C. followed by 1 minute at 80°C. Samples were set up using two different PCR buffers and all sampleswere set up as triplicates.

To evaluate how ExoSAP-IT would perform given a protocol identical toHL-ExoI, samples were spiked with reverse primers before the addition of2 μl of the ExoSAP-IT PCR clean-up reagent. Samples were set up asduplicates.

As negative controls, samples received reverse primers or dNTPs, butinstead of enzymatic clean-up solutions, samples received 2 μl water.Samples were set up as duplicates.

TABLE 2 provides an overview of the above described experimental set up.Reagent Volume Post-PCR solution (TEMPase Extra, VWR or GoTaq, Promega)4 μl Spike dNTP (10 mM ACGT each) or Reverse primer (10 μM) 1 μl PCRclean-up ExoSAP-IT (commercially available PCR clean-up solution) or 2μl HL-Exol & SAP or dH₂O Total 7 μl

TABLE 3 provides an overview of the PCR clean-up incubation ProtocolIncubation Total time ExoSAP-IT 15 minutes at 37° C. → 15 minutes at 80°C. 15 minutes 4 minutes at 37° C. → 1 minute at 80° C. 5 minutesHL-Exol + 4 minutes at 37° C. → 1 minute at 80° C. 5 minutes SAPNegative 15 minutes at 37° C. → 15 minutes at 80° C. 15 minutes control

Following PCR clean-up treatment, samples were immediately cooled onice. A prepared sequencing reaction mastermix was aliquoted intoseparate tubes. A total of 2.5 μl of each treated/untreated solution wasadded to each tube to be used as a template in the subsequent sequencingreaction. Samples were immediately transferred to a thermocycler and thesequencing program was initiated.

Reagent Volume BigDye v3.1 (LifeTech) 1 μl 5X Sequencing Buffer(LifeTech) 4 μl Sequencing primer (10 μM) 0.32 μl Template(treated/untreated PCR product with spike) 2.5 μl dH₂O 12.18 μl Total 20μl

Cycling temperature Time 96° C. 5 min 96° C. 10 sec 50° C. 5 sec {closeoversize bracket} 25 cycles 60° C. 4 min  4° C. Hold

Sequences were delivered to the DNA Sequencing core Facility atUniversity of Tromso for purification and sequencing using an AppliedBiosystems 3130xl Genetic Analyzer. Results were analyzed using theSequence Scanner Software v1.0 (LifeTech).

Selected results are shown in FIG. 14. Sequences spiked with dNTPyielded an overall good sequence length and quality, and no differencebetween samples treated with ExoSAP-IT or HL-ExoI/SAP could be detected(results not shown). The sequence plots of FIG. 14A are examples of theresults from sequences spiked with reverse primers in the GoTaq PCRbuffer. It was evident from the negative controls that lack offunctional PCR clean-up strongly compromised the length and quality ofthe sequence. Samples treated with either ExoSAP-IT (30 minutesprotocol) or HL-ExoI/SAP (5 minutes protocol) showed very good sequencequality. These images were representative for all replicates.

The sequence plots of FIG. 14B are examples of the results fromsequences spiked with reverse primers in the TEMPase Extra PCR buffer.Samples with added reverse primers showed very good sequence length andquality upon treatment with either of the PCR clean-up solutions. Therewere no significant differences between the ExoSAP-IT-treated samples(30 minutes protocol) or the HL-ExoI/SAP-treated samples (5 minutesprotocol). Lack of PCR clean-up treatment resulted in shorter sequencesof lower quality. These images were representative for all replicates.

On the other hand FIG. 14C illustrates how the ExoSAP-IT clean-upsolution performed when having to perform the same 5 minutes protocol asHL-ExoI/SAP-protocol. Evident from the Figure is that treatment withExoSAP-IT did not result in sequences of high quality. This is likelydue to a combination of insufficient degradation of added primers aswell as residual ExoI activity degrading sequencing primers. It islikely that using a reaction set-up at room temperature would furthercompromise these results due to the residual ExoI activity degradingsequencing primers. Samples treated with HL-ExoI/SAP showed overallexcellent sequence length and quality.

Example 5—Inactivation Experiments to Determine Minimum InactivationTime and Temperature for Certain Heat-Labile Exonucleases of theInvention

Minimum inactivation temperature and time was determined for eachHL-ExoI under test under the given assay conditions. This was achievedby incubating HL-ExoI at different temperatures for different timeintervals. Following heat-treatment, 5′ labeled single stranded DNA wasadded and degree of substrate degradation was visualized (FIG. 15). Theamount of substrate degradation was compared to the results from FIG.16, and residual activity following heat treatment was estimated.

Undiluted HL-ExoI was added to Reaction Buffer (10 mM Tris-HCl, pH 8.5at 25° C., 50 mM KCl, 5 mM MgCl₂), giving a final volume of 9 μl.Samples were incubated for 5 minutes, 10 minutes or 15 minutes at 40°C., 45° C., 50° C. or 55° C. or for 5 minutes or 10 minutes at 60° C.Following cooling of samples, 5 pmol of a 5′FAM labeled oligonucleotide(GCTAACTACCACCTGATTAC; SEQ ID No 21) was added. Samples were furtherincubated at 30° C. for 30 minutes and then for 2 minutes at 95° C. ATBE-Urea Sample Buffer (Bio-Rad) was added and samples were applied to aprecast 20% acrylamide/7 M urea gel and run at 180 V for approximately45 minutes. All reagents were kept on ice during the full protocol andworkflow was performed on cooling blocks unless specified otherwise.PAGE results were imaged using the Molecular Imager PharosFX system(Bio-Rad).

Results are shown in FIG. 15 and indicate that various degrees ofsubstrate degradation are observed depending on temperature andtime-interval for heat-incubation. Overall, none of the HL-ExoI showedany signs of substrate degradation following incubation at 55° C. for 10minutes or more. Incubations of all of the HL-ExoI at 55° C. for 5minutes or 50° C. for 10 minutes showed essentially no, or at mostbetween 1 and 10%, substrate degradation.

Example 6—Determination of Sensitivity Threshold for InactivationExperiments by Measuring Degree of Substrate Degradation for a DilutionSeries of Exonucleases

In order to determine the minimum inactivation temperature for thevarious HL-ExoI of the previous Example, the sensitivity threshold forthe inactivation assay of Example 5 was determined. A semi-quantitativeassay was prepared using serial dilutions of the exonucleases andestimating the degree of substrate degradation for each dilution. Forcomparative measurements, the same assay conditions and reaction set-upas used in the inactivation assays were also applied here.

Each HL-ExoI under test was diluted 1, 10, 100, 200, 1000 and 10,000times, corresponding to 100%, 10%, 1%, 0.5%, 0.1% and 0.01% activities.The same buffer as used as Reaction Buffer was also used as DilutionBuffer (10 mM Tris-HCl, pH 8.5 at 25° C., 50 mM KCl, 5 mM MgCl₂).Reaction Buffer and 5 pmol of FAM-labeled substrate(GCTAACTACCACCTGATTAC; SEQ ID No 21) were premixed prior to HL-ExoIaddition. Total volume of reaction was 10 μl. Samples were incubated for30 minutes at 30° C., followed by 2 minutes at 95° C. A TBE-Urea SampleBuffer (Bio-Rad) was added and samples were applied to a precast 20%acrylamide/7M urea gel and run at 180 V for approx. 45 minutes. Allreagents were kept on ice during the full protocol and workflow wasperformed on cooling blocks unless specified otherwise. PAGE resultswere imaged using the Molecular Imager PharosFX system (Bio-Rad).

Results are shown in FIG. 16 and indicate that activity of theexonucleases could be detected to approximately 1% activity in the givenassay conditions.

Example 7—Assay for Determining Double-Stranded and Single-StrandedExonuclease Activity

Exonuclease activity is measured by incubating the test exonucleaseenzyme with a short 5′-FAM-DNA-TAMRA-3′ labelled single or doublestranded substrate of approximately 20 nucleotides. If the exonucleaseis able to degrade the substrate this will start immediately and thefluorophore will be released. The activity can be followed over timesince the released fluorophore can re-emit light upon light excitation.Specifically, the assay mix consists of 1 μl 10 μM ssDNA/dsDNA, 10 μl5×TDB (250 mM Tris-HCl, pH 8.5 at 25° C., 5 mM DTT, 1 mg/ml BSA, 10%Glycerol) and 29 μl MiliQ H₂O. 40 μl assay mix is transferred to thewells of a black flat bottom 96-well plate and 5 μl MiliQ H₂O ordilution buffer (as negative control) and enzyme samples are added tothe wells. The reactions are initiated by adding 5 μl 50 mM MgCl₂ usinga multichannel pipette, making the final volume of the reaction 50 μl.Fluorescence is measured immediately (excitation 485 nm and emission at520 nm) and then at appropriate time intervals, include a shaking step,and the reactions are allowed to proceed for 15 minutes. An increase influorescence indicates degradation of the substrate is taking place.

Example 8—Assay for Quantifying Single-Stranded Exonuclease Activity

Single strand DNA exonuclease activity was measured by incubating theenzyme with a denatured ³H-dATP incorporated PCR product. If theexonuclease is able to degrade the substrate the exonuclease willrelease acid soluble nucleotides that can be detected in a scintillationcounter. Excess high molecular weight substrate DNA is precipitated withtrichloroacetic acid (TCA). In this assay, one Unit (1 U) is defined asthe amount of enzyme that will catalyse the release of 10 nmolacid-soluble nucleotides in a final volume of 20 μl in 30 minutes at 30°C.

Specifically, the assay mix consisted of 4 μl 5× Exonuclease buffer (250mM Tris-HCl, pH 7.5 at 25° C., 50 mM MgCl₂, 5 mM DTT), 5 μl denaturedsubstrate (denatured by incubation for 3 minutes at 100° C. andimmediate transfer to an ice water bath for 3 minutes) and 6 μl MiliQH₂O. 15 μl was transferred to 1.5 ml microcentrifuge tubes on ice. Theenzyme under test was diluted when necessary and 5 μl each of enzymesample, control and blank was added to the assay mix and mixed bypipetting up and down. The samples were incubated in a water bath at 30°C. for 10 minutes. After incubation the reactions were placed on ice and20 μl ice cold calf thymus DNA (1 mg/ml) and 250 μl ice cold 10% (w/v)TCA were added immediately. The samples were then incubated on ice for15 minutes and centrifuged at 4° C. for 10 minutes at 13,000 rpm. Thesupernatants, 200 μl, were transferred to a 24-well plate and 0.8 mlUltima Gold XR Scintillation fluid was then added. The wells were sealedwith sealing tape and the samples were mixed thoroughly by shaking. Thesamples were counted in a MicroBeta² Plate Counter for 5 minutes.

Example 9—Assay for Quantifying Double-Stranded Exonuclease Activity

Double strand DNA exonuclease activity is measured in the same way asExample 8 with the exception that the PCR substrate is not denaturedprior to incubation with the enzyme.

Example 10—Comparison of the Double Stranded and Single StrandedExonuclease Activities of the Heat Labile Exonucleases of the Invention

Single strand DNA exonuclease activity was measured by incubating thetest enzyme with a denatured ³H-dATP incorporated PCR product. Doublestrand DNA exonuclease activity was similarly measured with theexception that the enzyme was incubated with a non-denatured ³H-dATPincorporated PCR product. If an exonuclease is able to degrade thesubstrate the exonuclease will release acid soluble nucleotides that canbe detected in a scintillation counter. Excess high molecular weightsubstrate DNA is precipitated with trichloroacetic acid (TCA). In thisassay, one Unit (1 U) is defined as the amount of enzyme that willcatalyse the release of 10 nmol acid-soluble nucleotides in a finalvolume of 20 μl in 30 minutes at 30° C. Specifically, the assay mixconsisted of 4 μl 5× buffer (50 mM Tris-HCl, pH 8.5 at 25° C., 250 mMKCl and 25 mM MgCl₂), 5 μl substrate (to obtain ssDNA the dsDNAsubstrate was denatured by incubation for 3 minutes at 100° C. andimmediate transferred to an ice water bath for 3 minutes) and 3 μl MiliQH₂O. The assay mix, 12 μl, was transferred to 1.5 ml microcentrifugetubes on ice. The enzyme under test was diluted and 8 μl of enzymesample was added to the assay mix and mixed by pipetting up and down.Control and blank were also included in the set-up. The samples wereincubated in a water bath at 30° C. for 10 minutes. After incubation thereactions were placed on ice and 20 μl ice cold calf thymus DNA (1mg/ml) and 250 μl ice cold 10% (w/v) TCA were added immediately. Thesamples were then incubated on ice for 15 minutes and centrifuged at 4°C. for 10 minutes at 13,000 rpm. The supernatants, 200 μl, weretransferred to a 24-well plate and added 0.8 ml Ultima Gold XRScintillation fluid. The wells were sealed with sealing tape and thesamples mixed thoroughly by shaking. The samples were counted in aMicroBeta² Plate Counter for 5 minutes.

The results are summarized in Table 4 and shows that the HL-ExoI of theinvention display very little activity against double stranded DNAcompared to the activity against single stranded DNA. It is howeverdifficult to conclude if the low amounts of activity against doublestranded DNA are related to intrinsic properties of the enzymes or iscaused merely by contaminations of the enzymes. The two commercial ExoI(ExoI A and ExoI B) tested also displayed very low activity againstdouble stranded DNA.

TABLE 4 Activity of HL-Exol and commercial Exoi against ssDNA and dsDNA.Relative activity Activity Activity against dsDNA against ssDNA againstdsDNA and ssDNA (U/μl) (U/μl) (%) HL-Exol Ha 50.9 0.05 0.10 HL-Exol Ps11.2 0.02 0.18 HL-Exol Sh 65.1 0.01 0.02 HL-Exol Vw 21.0 0.01 0.05HL-Exol Mv 16.7 0.01 0.06 Exol A 16.7 0.01 0.06 Exol B 41.2 0.01 0.02

Example 11—Activity Profiling of Exonucleases: DirectionalityDetermination Using Urea Polyacrylamide Gel

This experiment was performed in order to verify that the HL-ExoI of theinvention exhibited 3′ to 5′ exonuclease activity and no substantial 5′to 3′ exonuclease activity. The substrate specificity was analysed usingeither a 5′ FAM or a 3′ FAM-labeled oligonucleotide(GCTAACTACCACCTGATTAC; SEQ ID No 21) on a polyacrylamide gel.

Each HL-ExoI under test was diluted to a final concentration of about0.1 U/μl. Two master mixes were prepared, one for the 5′ FAM-labeledoligo and one for the 3′ FAM-labeled oligo. The master mix containedReaction Buffer (10 mM Tris pH 8.5, 50 mM KCl, 5 mM MgCl₂) and therespective FAM-labeled substrate, giving a final amount of 0.25 pmolsubstrate per reaction. Both a negative control, containing dilutionbuffer instead of enzyme solution, and a positive control, using Exo Ifrom E. coli, were included. The enzymes were pipetted into thepre-cooled reaction tubes and then the master mix with the reactionbuffer and substrate was added. All reactions consisted of a finalvolume of 10 μl and were incubated at 30° C. for 1 h. Reactions werestopped by adding 2.5 μl of sample loading buffer (95% formamide, 10 mMEDTA, Xylene) and incubated at 95° C. for 5 min. For analysis, 6 μl ofthe samples were loaded onto a 12% acrylamide/7 M urea gel. The gel wasrun at 50 W for 1 h 45 minutes. During the set-up of the reactions allreagents and samples were kept on ice.

Results are shown in FIG. 17. Clear differences between the 5′FAM-labeled and 3′ FAM-labeled substrate are observed, indicating 3′ to5′ directionality of the different HL-ExoI as well as for the E. coliExoI control. When FAM was labeled at the 5′ end, a ladder of partialfaint and intense intermediate product bands were seen indicating theExoI degrading the substrate from the 3′ end. When the oligo wasFAM-labeled at the 3′ end, the fluorophore was immediately cut off,generating only the 3′FAM monomer.

Example 12—Activity Profiling of Exonucleases: Directionality onCrystallisation Structure

To further support the directionality of the HL-ExoI I enzymes of theinvention having 3′-5′ directionality, the HL-ExoI (Mv) was crystalisedwith ssDNA and the structure of the complex was determined.

Protein crystallisation was performed with a protein concentration of5.4 mg/ml. The desalted 13mer oligonucleotide (dT13) was purchased fromSigma Aldrich and added to the protein in a 1.2 molar excess. To inhibitthe degradation of the ssDNA by the exonucleaseI, 10 mM EDTA was added.The drops were set up automatically in a 96-well format in MRC 2 WellCrystallization Plates (Swissci, Hampton Research) with the Phenix (ArtRobbin Instruments) using the sitting drop vapor diffusion technique.The drop size was 0.4 μl (0.2 μl+0.2 μl) and the volume of the reservoirsolution was 60 μl. The crystallisation plates were incubated at 4° C.

A crystal co-crystallised with dT13/EDTA grew with 20.02% PEG MME 5000,0.1 M Na-acetate pH 4.5 and 0.09 M Ca-acetate. The X-ray diffractionexperiment was performed at the ESRF in Grenoble (France). The crystaldiffracted to 2.5 Å resolution. Structure determination has beenperformed by molecular replacement with the E. coli ExoI (PDB: 1FXX) asthe search model.

In the early rounds of refinement, electron density for the ssDNA becamevisible and all nucleotides could be fit in. The ssDNA binds with the3′-end in the active site in a similar manner as seen for E. coli Exo I(Korada et al., Nucleic Acids Research, 2013, 41(11):5887-97) providingstructural evidence for the HL-ExoI (Mv) 3′-5′directionality.

Example 13—Activity Profiling of Exonucleases: Rapid Inactivation ofCertain HL-ExoI of the Invention at 80° C.

This experiment was performed to confirm that certain of the HL-ExoI ofthe invention could perform satisfactorily in a rapid PCR clean-upscenario. In this experiment, the thermal inactivation characteristicsof the HL-ExoI under test at 80° C. were compared to two commerciallyavailable E. coli ExoI (ExoI A and ExoI B).

The activities of the HL-ExoI of the invention against ssDNA wascalculated as described in Example 8, with the exception that the 1×assay mix consisted of 67 mM Glycin-KOH, pH 9.5, 63.5 mM NaCl, 9.2 mMMgCl₂, 10 mM DTT including ³H dA-labelled DNA. For the commercial E.coli ExoI, the activity was taken as that stated by the manufacturers.To mimic the set-up in a PCR clean-up setting, a post PCR buffer wasused as the reaction buffer. The composition of the reaction buffer was10 mM Tris-HCl pH 8.5 (25° C.), 50 mM KCl, 1.5 mM MgCl₂, DyNAzyme II(Thermo Fisher Scientific™, formerly Finnzymes™) and remnants of dNTPs(initially 200 μM of each before the PCR-reaction was run), remnants ofprimers (initially 200 nM of each primer before the PCR-reaction wasrun) and template.

Each reaction received 10 U ExoI, giving a final reaction volume of 7μl. The experiment contained both an activity control for all ExoI, aswell as a check for residual activity following heat incubation. For theactivity control, reaction buffer, ExoI and 5 pmol substrate(GCTAACTACCACCTGATTAC; SEQ ID No 21) were mixed and incubated for 15minutes at 30° C. followed by 95° C. for 20 minutes. For samples to beanalysed with respect to inactivation at 80° C., substrate was addedfollowing 1 minute incubation at 80° C. and subsequent cooling. Thesesamples were subsequently incubated for 15 minutes at 30° C. followed by95° C. for 20 minutes. Following all incubation steps, TBE-Urea Samplebuffer was added and samples were applied to a precast 20% acrylamide/7M urea gel and run at 180 V for approximately 45 minutes.

Results are shown in FIG. 19. All Exo I had adequate activity in thisassay. Only the HL-ExoI were inactivated following 1 minute incubationat 80° C., while the two commercially available E. coli Exo I showedadequate residual activity degrading 100% of the substrate. The HL-ExoI,but not the commercial ExoI, compatible with a rapid 5 minute PCRclean-up protocol.

To further compare the ease of inactivation of HL-ExoI (Sh) with that oftwo commercially available E. coli ExoI, 10 U of each ExoI was incubatedfor 1, 5, 10 or 20 minutes at 80° C. before cooling and addition of 5pmol of a 5′ FAM labeled substrate (GCTAACTACCACCTGATTAC; SEQ ID No 21).The samples were further incubated for 15 minutes at 30° C. followed by20 minutes at 95° C. Reaction set-up was otherwise identical to theabove experiment, using the same post-PCR buffer as reaction buffer. Anactivity control was included for the three ExoI, and these samples werenot subjected to heat incubation prior to incubation at 30° C. for 15minutes. Residual activity was visualized on a Urea-PAGE gel asdescribed above.

Results are shown in FIG. 20. Substantial residual activity was observedin both commercially available ExoI following heat treatment at 80° C.for nearly all time durations. In comparison, no residual activity couldbe detected in the samples treated with HL-ExoI (Sh) which had beentreated at 80° C. for even a single minute.

Example 14—Demonstration of the Utility of HL-ExoI in a Rapid One-TubePCR Clean-Up Prior to Nucleic Acid Sequencing

This example was performed to show functionality of certain HL-ExoI ofthe invention in a PCR clean-up situation. The experimental set-up wasvery similar to Experiment 4. As in Example 4, to the post PCR solutionunder test was added excess primers (10 pmol) following the PCR.However, unlike Example 4 only one PCR buffer (GoTaq, Promega) was used,the incubation temperature for samples treated with HL-ExoI was reducedfrom 37° C. to 30° C. and only the regular 30 minutes protocol wastested for ExoSAP-IT.

Prior to initiating the experiment, the activity of each HL-ExoI wascalculated as described in Example 8, with the exception that the 1×assay mix consisted of 67 mM Glycin-KOH, pH 9.5, 63.5 mM NaCl, 9.2 mMMgCl₂, 10 mM DTT including ³H dA-labelled DNA. Samples treated withHL-ExoI received the amount of primers as stated above, before theaddition of 2 μl premixed HL-ExoI (10-20 U/μl) and SAP (1.5 U/μl). Totalvolume for each clean-up reaction was 7 μl. Samples were incubated 4minutes at 30° C. followed by 1 minute at 80° C. Samples were set up astriplicates

For comparison and positive control, samples were treated with a leadingbrand of enzymatic PCR clean-up; ExoSAP-IT™ (Affymetrix™). Samplestreated with ExoSAP-IT were handled according to manufacturer protocol.Following addition of primers to the PCR solution, samples received 2 μlof the PCR clean-up reagent, giving a final volume of 7 μl.ExoSAP-IT-treated samples were incubated 15 minutes at 37° C. followedby 15 minutes at 80° C. Samples were set up as triplicates.

Negative controls were set up and these received the same amount ofprimers as treated samples. Instead of enzymatic clean-up solution,these samples received 2 μl water. Samples were set up as triplicates.

Example 4 should be referred to for more details.

Sequences were delivered to the DNA Sequencing core Facility atUniversity of Tromso for purification and sequencing using AppliedBiosystems 3500xl Genetic Analyzer. Results were analyzed using theSequence Scanner Software v2.0 (LifeTech)

Selected results are shown in FIG. 21, where the sequence plots arerepresentative examples of the results from sequences spiked withreverse primers in the GoTaq PCR buffer. It was evident from thenegative controls that lack of functional PCR clean-up stronglycompromised the length and quality of the sequence. Samples treated witheither ExoSAP-IT (30 minutes protocol) or HL-ExoI/SAP (5 minutesprotocol) showed very good sequence quality.

1. A method of nucleic acid amplification, said method comprising astep, subsequent to the final amplification step, of contacting theproduct of the nucleic acid amplification reaction with an exonucleaseor enzymatically active fragment thereof under conditions which permitthe digestion of at least a portion of any single stranded DNA presentin the product and optionally then heating the mixture to inactivatesaid exonuclease or enzymatically active fragment thereof, wherein saidexonuclease has the amino acid sequence of SEQ ID No. 1 or an amino acidsequence which is at least about 85% identical thereto, wherein saidenzymatically active fragment of said exonuclease is at least about 85%identical to the amino acid sequence of said exonuclease, wherein saidexonuclease or enzymatically active fragment thereof (i) is at least 90%irreversibly inactivated by heating at a temperature of about 55° C. for10 mins in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50mM KCl and 5 mM MgCl₂; (ii) has activity against double stranded DNAthat is equal to or less than 15% of its activity against an equivalentamount of single stranded DNA under the same assay conditions; and (iii)has a 3′-5′ exonuclease activity.
 2. The method of claim 1, wherein saidmethod further comprises, subsequent to the final amplification step,contacting an alkaline phosphatase with the product of the amplificationreaction prior to, at the same time as, or after, contacting saidproduct with the exonuclease or enzymatically active fragment thereof,under conditions which permit the dephosphorylation of anyunincorporated nucleotide triphosphates present in the product.
 3. Themethod of claim 1, wherein said product is the direct product of thefinal amplification step.
 4. The method of claim 1, wherein said nucleicacid amplification reaction is selected from PCR, 3SR, SDA, LAR/LCR, andLAMP.
 5. A method of reverse transcription, said method comprising astep, subsequent to the final reverse transcription step, and/or, ifpresent, the final second strand cDNA synthesis step, of contacting theproduct of the reverse transcription reaction with an exonuclease orenzymatically active fragment thereof under conditions which permit thedigestion of at least a portion of any single stranded DNA present inthe product and optionally then heating the mixture to inactivate saidexonuclease or enzymatically active fragment thereof, wherein saidexonuclease has the amino acid sequence of SEQ ID No. 1 or an amino acidsequence which is at least about 85% identical thereto, wherein saidenzymatically active fragment of said exonuclease is at least about 85%identical to the amino acid sequence of said exonuclease, wherein saidexonuclease or enzymatically active fragment thereof (i) is at least 90%irreversibly inactivated by heating at a temperature of about 55° C. for10 mins in a buffer consisting of 10 mM Tris-HCl, pH 8.5 at 25° C., 50mM KCl and 5 mM MgCl₂; (ii) has activity against double stranded DNAthat is equal to or less than 15% of its activity against an equivalentamount of single stranded DNA under the same assay conditions; and (iii)has a 3′-5′ exonuclease activity.
 6. The method of claim 5, wherein saidmethod further comprises, subsequent to the final reverse transcriptionstep, and/or, if present, the final second strand cDNA synthesis step,contacting an alkaline phosphatase with the product of the reversetranscription reaction prior to, at the same time as, or after,contacting said product with the exonuclease or enzymatically activefragment thereof, under conditions which permit the dephosphorylation ofany unincorporated nucleotide triphosphates present in the product. 7.The method of claim 5, wherein said product is the direct product of thefinal reverse transcription step, and/or, if present, the direct productof the final second strand cDNA synthesis step.
 8. A method of nucleicacid sequence analysis, said method comprising a step of samplepreparation prior to the analysis step(s), said step of samplepreparation comprising contacting the sample to be analysed with anexonuclease or enzymatically active fragment thereof under conditionswhich permit the digestion of at least a portion of any single strandedDNA present in the sample and optionally then heating the mixture toinactivate said exonuclease or enzymatically active fragment thereof,wherein said exonuclease has the amino acid sequence of SEQ ID No. 1 oran amino acid sequence which is at least about 85% identical thereto,wherein said enzymatically active fragment of said exonuclease is atleast about 85% identical to the amino acid sequence of saidexonuclease, wherein said exonuclease or enzymatically active fragmentthereof (i) is at least 90% irreversibly inactivated by heating at atemperature of about 55° C. for 10 mins in a buffer consisting of 10 mMTris-HCl, pH 8.5 at 25° C., 50 mM KCl and 5 mM MgCl₂; (ii) has activityagainst double stranded DNA that is equal to or less than 15% of itsactivity against an equivalent amount of single stranded DNA under thesame assay conditions; and (iii) has a 3′-5′ exonuclease activity. 9.The method of claim 8, wherein said step of sample preparation furthercomprises contacting an alkaline phosphatase with the sample to beanalysed prior to, at the same time as, or after, contacting the samplewith the exonuclease or enzymatically active fragment thereof, underconditions which permit the dephosphorylation of any unincorporatednucleotide triphosphates present in the sample.
 10. The method of claim8, wherein said nucleic acid sequence analysis is a sequencing techniqueor an oligonucleotide hybridisation probe based technique.
 11. Themethod as claimed in claim 1, wherein said exonuclease has the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO:
 11. 12. The method asclaimed in claim 2, wherein said exonuclease has the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO:
 11. 13. The method as claimed in claim 3,wherein said exonuclease has the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO:
 11. 14. The method as claimed in claim 4, wherein saidexonuclease has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:11.
 15. The method as claimed in claim 5, wherein said exonuclease hasthe amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:
 11. 16. The methodas claimed in claim 6, wherein said exonuclease has the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO:
 11. 17. The method as claimed inclaim 7, wherein said exonuclease has the amino acid sequence of SEQ IDNO: 1 or SEQ ID NO:
 11. 18. The method as claimed in claim 8, whereinsaid exonuclease has the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO:
 11. 19. The method as claimed in claim 9, wherein said exonucleasehas the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:
 11. 20. Themethod as claimed in claim 10, wherein said exonuclease has the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.